This study will examine molecular mechanisms involved in regulating temporal expression of members of the lactate dehydrogenase (LDH) multigene family during spermatogenesis. The basic assumption is that the regulation of LDH gene expression in mouse germ cells during development results from a series of complex cooperative LDH promoter DNA-protein and protein-protein interactions. Published sequence information available for the 5' untranslated regions of the mouse LDH/B and LDH/C CDNAS will be used in this study to design a nested set of gene-specific primers. These primers will facilitate isolation of the LDH/B and LDH/C 5' upstream promoter/enhancer regions utilizing both traditional genomic library screening and an innovative PCR based "Genomic Walking" strategy. Stage-specific patterns of regulatory protein binding sites on the respective LDH promoter/enhancer elements will be analyzed using a combination of in vivo genomic footprinting, sequencing and methylation assays. Comparative studies utilizing in vitro DNAse I footprinting, Gel Shift mobility and Southwestern assays will be utilized to define additional functional interactions between these regulatory proteins. Ligand mediated expression screening techniques will be used to isolate and characterize CDNA clones corresponding to transcripts encoding these regulatory DNA binding proteins. Isolation and characterization of transacting factors regulating developmental and tissue specific expression of the LDH multigene family are expected to make significant contributions to our long term goals of understanding molecular mechanisms that control cellular proliferation and differentiation in mammalian germ cells.